crude bacterial DNA prep

Deanne Bell dbell at fresno.ars.usda.gov
Tue Aug 9 13:40:22 EST 2005


Hey Michael

For PCRs I regularly use bacteria that have been scraped from media
plates, suspended in water, aliquoted into utubes and stored in the
freezer.  Just thaw an aliquote and use 1ul at 10e8 cells/ml in a 25ul
PCR.  I would go ahead and try a PCR of I were you.  It is sooo much
easier than doing the DNA prep.

I have also been using this really easy prep protocol published by Blake
Bextine / Jacqueline Fletcher's lab using QBiogene's geneclean kits.  I
believe the reference is something about yellow vine disease and
associated bacterial infections of squash.  Plant Diseases 2001. Let me
know if you want my protocol.

Deanne Bell
USDA Agricultural Research Service
Crop Diseases, Pests & Genetics
9611 South Riverbend Avenue
Parlier, CA  93648
voice  (559) 596-2806
fax      (559) 596-2897
dbell at fresno.ars.usda.gov
 
 
 

>-----Original Message-----
>From: methods-bounces at oat.bio.indiana.edu 
>[mailto:methods-bounces at oat.bio.indiana.edu] On Behalf Of 
>Michael Sullivan
>Sent: Monday, August 08, 2005 9:59 AM
>To: methods at magpie.bio.indiana.edu
>Subject: crude bacterial DNA prep
>
>I want to use PCR to confirm the presence of a large, low copy 
>number plasmid from a bacterial strain.
>
>Can one simply use a small amount of culture directly in a PCR 
>reaction, or should one do some sort of DNA prep, even a crude 
>one, prior to PCR? If the latter, does anybody have any 
>suggestions for an easy prep to make PCR-quality DNA.
>
>Thanks,
>
>Mike
>---
>Michael L. Sullivan
>Plant Research Molecular Geneticist
>US Dairy Forage Research Center
>ARS-USDA
>1925 Linden Drive West
>Madison, WI 53706
>(608) 890-0046 (Phone)
>(608) 890-0076 (FAX)
>
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